Protease-regulated antibodies

ABSTRACT

This disclosure provides protease-regulated antibodies which specifically bind to tissue factor pathway inhibitor (TFPI). The antibodies are useful for treating bleeding disorders such as hemophilia.

This application claims the benefit of Ser. No. 61/617,837 filed on Mar.30, 2012, which is incorporated herein by reference in its entirety.

All documents cited in this disclosure are incorporated herein byreference in their entireties.

This application incorporates by reference a 64 kb text file created onMar. 26, 2012 and named “0297301274sequencelisting.txt,” which is thesequence listing for this application.

TECHNICAL HELD

The technical field is treatment of hemophilia and other coagulopathies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Illustration of an embodiment of a protease-regulated antibody.VI, heavy chain variable region; VT, light chain variable region; CH,heavy chain constant region; CL, light chain constant region,

FIG. 2. Western blot of two protease-regulated anti-TFPI Fab fragments(“Fab-1” and “Fab-2”), with and without thrombin digestion.

FIG. 3. Graph showing tissue factor pathway inhibitor (TFPI) binding ofFab-1 and Fab-2, with and without thrombin digestion, assayed by ELISA.

FIG. 4. Graph showing BIACORE™ measurement of TFPI binding of Fab-1(Fab1) and Fab-2 (Fab2) with and without thrombin digestion.

FIG. 5. SDS-PAGE of purified IgG antibody expressed in HEK293 6.E cells.

FIG. 6. Western Blot of parental IgG (“Parent IgG;” top) andIgG2-linker1 (bottom) Lane 1, digestion with thrombin (1 unit); lane 2,no protease (control); lane 3, digestion with bovine plasmin; lane 4,digestion with enterokinase (0.02 μg); lane 5, digestion with bovineFactor Xa (1 μg); lane 6, digestion with matriptase (MTSP) (0.5 μg);lane 7, digestion with urokinase (uPA) (0.25 μg) lane 8, digestion withhuman rhinovirus 3C protease (HRV 3C) (2 units).

FIG. 7. Graph showing TFPI binding of IgG with and without thrombindigestion, assayed by ELISA.

FIG. 8. Graph showing BIACORE™ measurement of TFPI binding of parentalIgG and IgG-linker1, with and without thrombin digestion.

FIG. 9. Western blot showing protease digestion of IgG-Linker1 and WTantibody with human proteases.

DETAILED DESCRIPTION

This disclosure provides protease-regulated antibodies whichspecifically bind to tissue factor pathway inhibitor (TFPI). Theantibodies are useful for treating bleeding disorders such ashemophilia. In some embodiments, protease-regulated anti-TFPI antibodiescan be cleaved by thrombin and/or plasmin. By initially inhibiting TFPI,such protease-regulated anti-TFPI antibodies promote the generation ofthrombin and/or plasmin, which in turn cleaves the antibodies andremoves or significantly reduces their binding activity to TFPI. Thisnegative feedback allows the antibodies to promote coagulation within asafe therapeutic window.

1. Protease-Regulated Anti-TFPI Antibodies

Protease-regulated antibodies disclosed herein specifically bind toTFPI; i.e. they bind to TFPI with an affinity that is higher (e.g., atleast two-fold higher) than their binding affinity for an irrelevantantigen (e.g., BSA, casein). The term “tissue factor pathway inhibitor”or “TFPI” as used herein refers to any variant, isoform and specieshomolog of human TFPI that is naturally expressed by cells.

In some embodiments, protease-regulated antibodies bind to TFPI with anaffinity of at least about 10⁵ M⁻¹ to about 10¹² M⁻¹ (e.g., 10⁵ M⁻¹,10^(5.5) M⁻¹, 10⁶ M⁻¹, 10^(5.6) M⁻¹, 10⁷ M⁻¹, 10^(7.5) M⁻¹, 10⁸ M⁻¹,10^(8.5) M⁻¹, 10⁹ M⁻¹, 10^(9.5) M⁻¹, 10¹⁰ M⁻¹, 10^(10.5) M⁻¹, 10¹¹ M⁻¹,10^(11.5) M⁻¹, 10¹² M⁻¹). The affinity (KO of antibody binding to anantigen can be assayed using any method known in the art including, forexample, immunoassays such as enzyme-linked immununospecific assay(ELISA), Bimolecular Interaction Analysis (BIA) (e.g., Sjolander &Urbaniczky; Anal. Chem. 63:2338-2345, 1991; Szabo, et al., Curr. Opin.Struct. Biol. 5:699-705, 1995), and fluorescence-activated cell sorting(FACS) for quantification of antibody binding to cells that express an.antigen. BIA is a technology for analyzing biospecific interactions inreal time, without labeling any of the interactants (e.g., BIACORE™),Changes in the optical phenomenon surface plasmon resonance (SPR) can beused as an indication of real-time reactions between biologicalmolecules.

A protease-regulated anti-TFPI antibody can be constructed using asubstantially full-length immunoglobulin molecule (e.g., IgG 1, IgG2a,IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA), an antigen binding fragmentthereof, such as a Fab or. F(ab′)₂, or a construct containing an antigenbinding site, such as a scFv, Fv, or diabody, which is capable ofspecific binding to TFPI. The term “antibody” also includes otherprotein scaffolds that are able to orient antibodycomplementarity-determining region (CDR) inserts into the same activebinding conformation as that found in natural antibodies such that thebinding to TFPI observed with these chimeric proteins is maintainedrelative to the TFPI binding activity of the natural antibody from whichthe CDRs were derived.

An “isolated antibody” as used herein is an antibody which issubstantially free of other antibodies having different antigenicspecificities (e.g., an isolated antibody that binds to TFPI issubstantially free of antibodies that bind antigens other than TFPI). Anisolated antibody that binds to an epitope, isoform, or variant of humanTFPI may, however, have cross-reactivity to other related antigens,e.g., from other species (e.g., TFPI species homologs). An isolatedantibody can be substantially free of other cellular material and/orchemicals.

The protease-regulated antibodies disclosed herein are engineered tocomprise a protease cleavage site recognized by one or more proteases,As used herein, “protease cleavage site” refers to an amino acidsequence that is recognized and cleaved by a. protease. In someembodiments, the protease cleavage site is positioned between itsvariable and constant regions. In some embodiments, protease-regulatedanti-TFPI antibodies include one or more protease cleavage sites thatcan be cleaved by thrombin, plasmin, and/or Factor Xa. In someembodiments, the amino acid sequence between the variable and constantregions of a protease-regulated anti-TFPI antibody comprises apolypeptide linker in addition to the protease cleavage site (asillustrated, for example, in FIG. 1). The linker can be a single aminoacid or a polypeptide sequence (e.g., up to 100 amino acids). Forexample, the tinker can be GGGGS (SEQ ID NO:149). Other useful linkersinclude those shown in SEQ ID NOS:151-176. In other embodiments, nolinker is present, and the cleavage site itself is inserted between thevariable and constant regions.

At least two optimal cleavage sites for thrombin have been determined:(1) X₁-X₂-P-R-X₃-X₄ (SEQ ID NO:147), Where X₁ and X₂ are hydrophobicamino acids and X₃ and X₄ are nonacidic amino acids; and (2) GRG.Thrombin specifically cleaves after the arginine residue. Plasmin canalso cleave the two aforementioned cleavage sites, however with lessspecificity as compared to thrombin. Other useful thrombin cleavagesites are provided as SEQ ID NOS:1-60. Other useful plasmin cleavagessites are provided as SEQ ID NOS:12, 47, 18, 53, and 61-130. In someembodiments, the cleavage site is LVPRGS (SEQ ID NO:137).

In some embodiments, a Factor Xa cleavage site, such as I-(E or D)-G-R(SEQ ID NO:148), is used. Other useful Factor Xa cleavage sites areprovided as SEQ ID NOS:29, 59, and 61-69. Other thrombin and FXacleavage sites or sequences can be found from previous publicationauthored by Bianchini [Bianchini E P et al 2002 :IBC]. Oneprotease-regulated antibody may comprise more than one protease cleavagesites.

In addition to cleavage site, a second binding site of protease,so-called exosite, can be introduced into a protease-regulated TFPIantibody to make the cleavage more efficient. The exosite of thrombincan be from the native exosite of protease substrates or inibitor, suchas PAR1, fibrinogen and hirudin. The exosite can also be a derivative ofnative exosite.

Protease-regulated TFPI antibodies can be produced synthetically orrecombinantly. A number of technologies are available to produceantibodies. For example, phage-antibody technology can be used togenerate antibodies (Knappik et al., J. Mol. Biol. 296:57-86, 2000),Another approach for obtaining antibodies is to screen a DNA libraryfrom B cells as described in WO 91/17271 and WO 92/01047. In thesemethods, libraries of phage are produced in which members displaydifferent antibodies on their outer surfaces. Antibodies are usuallydisplayed as Fv or Fab fragments. Phage displaying antibodies areselected by affinity enrichment for binding to a selected protein.Antibodies can also be produced using trioma methodology Oestberg etal., Hybridoma 2:361-367, 1983; U.S. Pat. No. 4,634,664; U.S. Pat. No.4,634,666).

Antibodies can also be purified from any cell that expresses theantibodies, including host cells that have been transfected withantibody-encoding expression constructs. The host cells can be culturedunder conditions whereby the antibodies are expressed. Purified antibodycan be separated from other cellular components that can associate withthe antibody in the cell, such as certain proteins, carbohydrates, orlipids, using methods well known in the art. Such methods include, butare not limited to, size exclusion chromatography, ammonium sulfatefractionation, ion exchange chromatography, affinity chromatography, andpreparative gel electrophoresis. Purity of the preparations can beassessed by any means known in the art, such as SDS-polyacrylamide gelelectrophoresis. A preparation of purified antibodies can contain morethan one type of antibody.

Alternatively, protease-regulated anti-TFPI antibodies can be producedusing chemical methods to synthesize its amino acid sequence, such as bydirect peptide synthesis using solid-phase techniques (e.g., Merrifield,J. Am. Chem. Soc. 85:2149-2154, 1963; Roberge et al., Science269:202-204, 1995). Protein synthesis can be performed using manualtechniques or by automation. Optionally, fragments of antibodies can beseparately synthesized and combined using chemical methods to produce afull-length molecule.

A protease-regulated anti-TFPI antibody can also be constructed in a“single chain PV (scFv) format,” in which a protease cleavage site isinserted in or around a peptide linker between the variable region oflight chain and the variable region of heavy chain of a scFv. As thepeptide linker is necessary to hold together the two variable regions ofa scFv for antigen binding, cleavage of the peptide linker or flankingregion allows a protease of interest to inactivate or to down-regulatethe binding of scFv to its antigen. The amino acid sequence SEQ IDNO:179 (which can be encoded by SEQ 11) NO:180) is an example of aprotease-regulated anti-TFPI antibodies in scFv format.

In some embodiments, protease-regulated TFPI antibodies are constructedin “IgG format,” having two binding sites, and can comprise one, two,three, or four protease cleavage sites on the heavy chain, light chain,or both. In each case, a protease cleavage site can be flanked on eitheror both sides by a linker. Further, in each case, the cleavage sites canbe the same or different. “IgG-linker1” (Example 5) and “IgG-linker2”are examples of protease-regulated anti-TFPI antibodies in IgG format:

light chain heavy chain amino acid DNA amino acid DNA IgG-linker1 SEQ IDSEQ ID SEQ ID SEQ ID NO: 139 NO: 141 NO: 140 NO: 142 IgG-linker2 SEQ IDSEQ ID SEQ ID SEQ ID NO: 143 NO: 145 NO: 144 NO: 146

2. Negative Feedback

Protease-regulated antibodies can include a protease cleavage site of aprotease upregulated or generated as a result of the function of theantibody. Such protease can then cleave the protease-regulated antibodythereby providing a negative feedback loop which can be useful inpreventing excess activity of the protease-regulated. antibody.

For example, protease-regulated anti-TFPI antibodies which can becleaved by thrombin and/or plasmin can exhibit such a negative feedbackeffect. Such protease-regulated anti-TFPI antibodies promote thegeneration of thrombin and/or plasmin. Thrombin and plasmin will in turncleave the protease-regulated anti-TFPI antibodies and remove orsignificantly reduce their binding activity to TFPI. This negativefeedback allows the antibodies to promote coagulation within a safetherapeutic window.

3. Polynucleotides

This disclosure also provides polynucleotides encodingprotease-regulated antibodies. These polynucleotides can be used, forexample, to produce quantities of the antibodies for therapeutic use.

Antibody-encoding cDNA molecules can be made with standard molecularbiology techniques, using mRNA as a template. Thereafter, cDNA moleculescan be replicated using molecular biology techniques known in the artand disclosed in manuals such as Sambrook, et al., (Molecular Cloning: ALaboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press;Cold Spring Harbor, N.Y.; 1989) Vol. 1-3). An amplification technique,such as PCR, can be used to obtain additional copies of thepolynucleotides. Alternatively, synthetic chemistry techniques can beused to synthesize polynucleotides encoding protease-regulated anti-TFPIantibodies.

To express a polynucleotide encoding an antibody, the polynucleotide canbe inserted into an expression vector that contains the necessaryelements for the transcription and translation of the inserted codingsequence. Methods that are well known to those skilled in the art can beused to construct expression vectors containing sequences encodingantibodies and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described, for example, in Sambrook, et al. (1989) and in Ausubel,et al., (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1995).

A variety of expression vector/host systems can be utilized to containand express sequences encoding antibodies. These include, but are notlimited to, microorganisms, such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with vines expression vectors e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g. cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV); or bacterial expressionvectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translatedregions of the vector enhancers, promoters, 5′ and 3′ untranslatedregions which interact with host cellular proteins to carry outtranscription and translation. Such elements can vary in strength andspecificity. Depending on the vector system and host, any number ofsuitable transcription and translation elements, including constitutiveand inducible promoters, can be used. For example, when cloning inbacterial systems, inducible promoters can be used. The baculoviruspolyhedrin promoter can be used in insect cells. Promoters or enhancersderived from the genomes of plant cells (e.g., heat shock, RUBISCO, andstorage protein genes) or from plant viruses (e.g., viral promoters orleader sequences) can be cloned into the vector. In mammalian cellsystems, promoters from mammalian genes or from mammalian viruses can beused. If it is necessary to generate a cell line that contains Multiplecopies of a nucleotide sequence encoding an antibody, vectors based onSV40 or EBV can be used with an appropriate selectable marker.

General texts describing additional useful molecular biologicaltechniques, including the preparation of antibodies, are Berger andKimmel (Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc.); Sambrook, et al., (Molecular Cloning: ALaboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press;Cold Spring Harbor, N.Y.; 1989) Vol. 1-3); Current Protocols inMolecular Biology, (F. M. Ausabel et al. [Eds.], Current Protocols, ajoint venture between Green Publishing Associates, Inc. and John Wiley &Sons, Inc. (supplemented through 2000)); Harlow et al., (MonoclonalAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1988), Paul [Ed.]); Fundamental Immunology, (Lippincott & Wilkins(1998)); and Harlow, et al. (Using Antibodies: A Laboratory Manual,Cold. Spring Harbor Laboratory Press (1998)).

4. Pharmaceutical Compositions

A protease-regulated anti-TFPI antibody can be provided in apharmaceutical composition comprising a pharmaceutically acceptablecarrier. The pharmaceutically acceptable carrier preferably isnon-pyrogenic. A pharmaceutical composition comprising aprotease-regulated anti-TFPI antibody can be administered alone or incombination with at least one other agent, such as stabilizing compound,which can be administered in any sterile, biocompatible pharmaceuticalcarrier, including, but not limited to, saline, buffered saline,dextrose, and water. A variety of aqueous carriers can be employed,e.g., 0.4% saline, 0.3% glycine, and the like. These solutions aresterile and generally free of particulate matter. These solutions can besterilized by conventional, well known sterilization techniques (e.g.,filtration). The compositions can contain pharmaceutically acceptableauxiliary substances as required to approximate physiological conditionssuch as pH adjusting and buffering agents, etc. The concentration ofprotease-regulated anti-TFPI antibody in a pharmaceutical compositioncan vary widely, i.e., from less than about 0.5%, usually at or at leastabout 1% to as much as 15 or 20% by weight and will be selectedprimarily based on fluid volumes, viscosities, etc., according to theparticular mode of administration selected. Sec U.S. Pat. No. 5,851,525.If desired, more than one different protease-regulated anti-TFPIantibody can be included in a pharmaceutical composition.

In addition to the active ingredients, pharmaceutical compositions cancontain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries that facilitate processing of thecompositions into preparations which can be used pharmaceutically.Pharmaceutical compositions can be administered by any number of routesincluding, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, parenteral,topical, sublingual, or rectal means.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. Such labeling would include amount, frequency, and method ofadministration.

5. Methods

Pharmaceutical compositions comprising one or more protease-regulatedanti-TFPI antibodies can be administered to a patient alone, or incombination with other agents, drugs or coagulation factors, to treathemophilia or other clotting disorders. A “therapeutically effectivedose” of protease-regulated anti-TFPI antibody refers to that amount ofprotease-regulated anti-TFPI antibody that will promote coagulation orreduce bleeding time. The determination of a therapeutically effectivedose is well within the capability of those skilled in the art.

A therapeutically effective dose can be estimated initially either incell culture assays or in animal models, usually rats, mice, rabbits,dogs, or pigs. An animal model also can be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeuticallyeffective in 50% of the population) and LD₅₀ (the dose lethal to 50% ofthe population) of a protease-regulated anti-TFPI antibody can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals. The dose ratio of toxic to therapeutic effects isthe therapeutic index, and it can be expressed as the ratio, LD₅₀/ED₅₀.

Pharmaceutical compositions that exhibit large therapeutic indices arepreferred. Data obtained from cell culture assays and animal studies areused in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the patient who requires treatment. Dosage andadministration are adjusted to provide sufficient levels of theprotease-regulated TFPI antibody or to maintain the desired effect.Factors that can be taken into account include the severity of thedisease state, general health of the subject, age, weight, and gender ofthe subject, diet, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long-acting pharmaceutical compositions can be administeredevery 3 to 4 days, every week, or once every two weeks depending on thehalf-life and clearance rate of the particular formulation.

In some embodiments, therapeutically effective in vivo dosages of aprotease-regulated anti-TFPI antibody are in the range of about 5 μg toabout 100 mg/kg, about 1 mg to about 50 mg/kg, about 10 mg to about 50mg/kg of patient body weight.

The mode of administration of a pharmaceutical composition comprising aprotease-regulated anti-TFPI antibody can be any suitable route whichdelivers the antibody to the host (e.g., subcutaneous, intramuscular,intravenous, or intranasal administration).

In some embodiments, a protease-regulated anti-TFPI antibody isadministered without other therapeutic agents. In some embodiments, aprotease-regulated anti-TFPI antibody is administered in combinationwith other agents, such as drugs or coagulation factors, to enhanceinitial production of thrombin while ensuring that the thrombin levelstays below the range that may cause thrombosis in some people withcoagulopathy. The administration of the protease-regulated anti-TFPIantibody can be before, after, or at substantially the same time as theadministration of other agents.

Nothing in this specification should be considered as limiting the scopeof this disclosure. All examples presented are representative andnon-limiting. The above-described embodiments can be modified or varied,as appreciated by those skilled in the art in light of the aboveteachings. It is therefore to be understood that, within the scope ofthe claims and their equivalents, the embodiments disclosed herein canbe practiced otherwise than as specifically described.

EXAMPLE 1 Construction of Protease-Regulated Anti-TFPI Fab Fragments

Two protease-regulated anti-TFPI Fabs, “Fab-1” and “Fab-2,” were basedon the anti-TFPI antibody sequences shown in SEQ ID NO: 177 (heavychain) and SEQ NO:178 (light chain). Both Fabs have the thrombin/plasminprotease cleavage. site, LVPRGS (SEQ ID NO:137) inserted C-terminal toboth variable domains. The cleavage site in Fab-1 is flanked by a(Gly)₄Ser linker. Fab-2 contains the six amino acid cleavage site alone,no linker is present. Thrombin and plasmin will cleave C-terminal to theArg (R) residue of LVPRGS (SEQ ID NO:137). DNA encoding the Fabs wassynthesized by GenScript with optimized codons for bacterial expression.The amino acid and DNA sequences for the Fabs are identified in thetable below.

Fab-1 variable and SEQ ID NO: 131 constant light amino acid sequenceFab-1 variable and SEQ ID NO: 133 constant heavy amino acid sequenceFab-1 DNA Sequence SEQ ID NO: 134 Fab-2 variable and SEQ ID NO: 135constant light amino acid sequence Fab-2 variable and SEQ ID NO: 136constant heavy amino acid sequence Fab-2 DNA Sequence SEQ ID NO: 138

The Fab coding regions were digested with the restriction enzymes BsaIand HindIII (New England Biolabs). The DNA fragments were purified usingan agarose gel and subcloned into pBADmycHisA (Invitrogen). The clonedDNA was ligated and transformed using standard techniques. Positiveclones were confirmed by DNA sequencing and used for BL21 E. coliexpression.

EXAMPLE 2 Western Blot of Thrombin-Cleaved Fabs

Approximately 2.5 μg of the crudely purified Fab-1 and Fah-2 weredigested with 0, 2 or 10 units of thrombin (Novagen) for 1 hr at 37° C.Digests were run on a 4-15% CRITEREON™ TGX™ (Bio-Rad). Protein weretransferred to a nitrocellulose membrane and probed with an anti-humanFab antibody (Southern Biotech).

The Western blot of Fab-1 and Fab-2 cleavage is shown in FIG. 2. Allsamples were reduced. Bands at approximately 12 kDA were observed forboth Fabs when treated with thrombin. These bands were not present inthe samples without thrombin digestion, indicating that the small sizeprotein was the product of thrombin cleavage.

EXAMPLE 3 TFPI ELISA of Thrombin Digested Fab-1 and Fab-2

Approximately 2.5 μg of partially purified Fab-1 and Fab-2 were digestedwith 2 units of biotinylated thrombin (Novagen) at 23° C. overnight.Streptavidin sepharose beads (100 μL) were added to the digestion todeplete the thrombin. The digested Fab samples were applied to a columnthat captures the sepharose bead/thrombin complex. The eluate containedthe digested Fabs.

A MAXISORP® 96-well plate (Nunc) was coated with 1 μg/mL of TFPI in PBSovernight at 4° C. The plate was blocked for 1 hr at room temperature(RT) in 5% non-fat dry milk PBS/0.5% TWEEN-20® (PBS-T). Serial two-folddilutions of undigested and digested Fab-1 and Fab-2 were added to thewells (100 μL/well) and incubated for 1 hr at RT. The plates were washedfive times with PBS-T. A secondary HRP-conjugated anti-Fab antibody wasadded (100 μL of a 1:10000 dilution) for detection with an AMPLEX® Red(Invitrogen) solution. As shown in FIG. 3, thrombin digestionsignificantly reduced the signal of TFPI binding.

EXAMPLE 4 BIACORE™ Measurements of Thrombin-Digested FAB-1 and FAB-2

Approximately 2.5 μg of crudely purified. Fab-1 and Fab-2 were digestedwith 2 units of biotinylated thrombin (Novagen) at 23° C. overnight.Streptavidin sepharose beads (100 μL) were added to the digestion todeplete the thrombin. The digested Fab samples were applied to a columnthat captures the sepharose bead/thrombin complex. The eluate containedthe digested Fabs.

For BIACORE™ analysis, human TFPI (American Diagnostica) was immobilizedusing amine coupling at targeted level of 100 relative units (RU), andthe antibodies were injected in the mobile phase. HBS-p was used as therunning buffer. A reference channel was prepared using blankimmobilization where the surface was activated and then inactivatedwithout any immobilized protein. A manual run was performed to measureelevated RU of the Fabs and a buffer control.

As shown in FIG. 4. The parental Fab without cleavable linker generated24.6 comparable to undigested Fab-1 (25.6 RU) and Fab2 (27.9 RU). Afterthrombin cleavage, the binding signal of Fab-1 and Fah-2 was reduced bymore than 50%, indicating that cleaved Fab-1 and Fab-2 not only lost theconstant domain, but also lost binding activity to TFPI.

EXAMPLE 5 IgG Expression and Purification

A protease-regulated anti-TFPI immunoglobulin molecules, “IgG-linker1,”was constructed based on parental anti-TFPI antibody sequences shown inSEQ ID NO:177 (heavy chain) and SEQ ID NO: 178 (light chain). Tofacilitate molecular cloning, a BlpI site was introduced in the heavychain coding sequence. Compared to the position of protease cleavablelinker in Fab-1, the introduction of BlpI site shifts the linkerposition of IgG-linker1 two amino acids to the constant region.

HEK293 6E cells were transfected with constructed IgG expressionvectors, and the culture supernatant containing the IgG antibodies washarvested. The antibodies were purified using an affinity column ofMABSELECT SURE™ followed by SUPERDEX™200 chromatography. The purifiedIgG-linker1 on SDS-PAGE is shown FIG. 5.

EXAMPLE 6 Western Blot of Parental IgG and IgG-Linker1

Purified parental IgG and IgG-linker1 (0.5 μg) were digested withthrombin, bovine plasmin, bovine Factor Xa, matriptase (MTSP), urokinase(uPA), or human rhinovirus 3C protease WWI 3C). The antibodies wereincubated with the proteases for 1 hr at 37° C. Digests were run on a4-20% CRITEREON™ TGX™ gel (Bio-Rad). Protein was transferred to anitrocellulose membrane and probed with an anti-human IgG heavy andlight chain antibody (Pierce). Western blots of IgG2 and IgG2-linker1are shown in FIG. 6. All samples were reduced.

Intact IgG produced two bands under reducing conditions. The two bandscorrespond to the heavy chain (50 kD) and the light chain (25 kD).Digested antibody IgG-linker1 showed a shift in molecular weight vs.undigested antibody. The following proteases were used to digests theantibodies: thrombin, plasmin, bovine Factor Xa, MTSP, and uPA. Thedigested IgG-linker1 antibody showed a shift in the molecular weight ofthe heavy chain from 50 kD to 37 kD. This size shift correlates with theloss of the VH domain from the heavy chain. There was also a molecularweight shift of the 25 kD light chain to a faint band around 16 kDa,which indicates cleavage of the VL domain from the light chain. Theproteases did not cleave parental IgG, indicating that molecular weightloss was a result of the protease digestion due to the cleavage siteengineered into the antibody.

EXAMPLE 7 TFPI-Binding ELISA of Thrombin Digested Parental IgG andIgG-Linker1

One microgram of the full length antibodies, parental IgG andIgG-linker1, were digested with 1 unit of biotinylated thrombin(Novagen) for 1 hr at 37° C. Then 50 μL of streptavidin sepharose beadswere added to the digestion to deplete the thrombin. The digested IgGsamples were applied to a column that captures the sepharosebead/thrombin complex. The dilate contains the digested IgGs.

A MAXISORP® 96-well plate (Nuns) was coated with 1 μg/mile of TFPI inPBS over night at 4° C. The plate was blocked for 1 hr at roomtemperature (RT) in 5% non-fat dry milk PBS/0.5% TWEEN® 20 (PBS-T).Serial two-fold dilutions of undigested and digested parental IgG andIgG-linker1 were added to the wells (100 μL/well) and incubated for 1 hrat RT. The plate was washed five times in PBS-T. A secondary HRPconjugated anti-Fab-antibody was added (100 μL of a 1:10,000 dilution)for detection with an AMPLEX RED® (Invitrogen) solution.

The results of the TFPI binding ELISA are shown in FIG. 7. Loss of TFPIbinding by thrombin digested IgG Linker-1 is shown. The TFPI binding ofundigested IgG-linker1 was similar to the TFPI binding of the undigestedand thrombin-digested parental IgG.

EXAMPLE 8 BIACORE™ Analysis of Parental IgG and IgG-Linker1

A CM4 sensor chip was immobilized with a low density of human TFPI usingan amine coupling kit (GE HealthCare). Kinetic assays of parental IgGand IgG-linker1 were conducted using different concentration of theantibodies, followed by regeneration with pH 1.5 glycine buffer. Theparental IgG and IgG-linker1 antibodies at a concentration of 1 μg weredigested with 1 unit of biotinylated thrombin (Novagen) for 1 hr at 37°C. The antibodies with or without digestion were injected to a BIACORE™system for TFPI-binding analysis. The following figure shows the signalgenerated from injection 45 μl of 6.25 μg/ml antibodies or controlsamples.

In kinetics assay, parental IgG and IgG2-linker1 have association rates(ka) of 1.536×10⁶/Ms and 1.902×10⁶/Ms, respectively. The two antibodiesdid not have measurable dissociation in 30 minutes. This indicates thatthe insertion of linker1 did not significantly change binding activityof the antibody.

The effect of thrombin cleavage on the antibodies is shown in FIG. S.Thrombin digestion slightly decreased the parental IgG binding on TFPI,whereas thrombin digestion reduced IgG2-linker1 binding on TFPI from 20RU to 5 RU, a 75% decrease.

EXAMPLE 9 Western Blot of Protease/Coagulation Factor TreatedIgG-Linker1 and Parental IgG

The following human proteases/coagulation factors were used to digest 80nM of IgG-Linker1 and the WT antibody: thrombin (0.1 μM), plasmin (0.1μM), Factor Vila (0.01 μM), Factor IXa (0.089 μM), Factor Xa (13 μM),Factor XIa (0.031 μM), Factor XIIIa (0.03 μM). The treated material wasrun on a 4-15% CRITEREON™ TGX gel (Bio-Rad). Proteins were transferredto a nitrocellulose membrane and probed with an anti-human IgG antibody(Pierce).

Human thrombin, plasmin and FactorXa digested IgG-Linker1, thrombindigesting most efficiently (FIG. 9) as shown by the appearance of a 37kDa band. The proteases did not cleave the parental IgG, indicating thatmolecular weight loss was a result of the protease digestion due to thecleavage site engineered into the antibody.

What is claimed is:
 1. A protease-regulated antibody which specificallybinds to tissue factor pathway inhibitor (TFPI), comprising a variabledomain, a constant domain, and an amino acid sequence joining thevariable domain to the constant domain, wherein the amino acid sequencecomprises a protease cleavage site.
 2. The protease-regulated antibodyof claim 1, wherein the protease cleavage site is a thrombin cleavagesite.
 3. The protease-regulated antibody of claim 1, wherein theprotease cleavage site is a plasmin cleavage site.
 4. Theprotease-regulated antibody of claim 1, wherein the protease cleavagesite is a Factor Xa cleavage site.
 5. The protease-regulated antibody ofclaim 1 which comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 131, amino acids 1-239 of SEQ ID NO: 133, SEQ:ID NO: 135, amino acids 1-229 of SEQ ID NO: 136, SEQ ID NO: 139, SEQ IDNO: 140, SEQ ID NO: 143, and SEQ ID NO:
 144. 6. The protease-regulatedantibody of claim 1, wherein the amino acid sequence further comprisesan amino acid linker.
 7. The protease-regulated antibody of claim 1,wherein the protease-regulated antibody is a full-length antibody. 8.The protease-regulated antibody of claim 1, wherein theprotease-regulated antibody is an antibody fragment.
 9. A pharmaceuticalcomposition comprising the protease-regulated antibody of claim 1, and apharmaceutically acceptable vehicle.
 10. An isolated nucleic acidmolecule encoding the protease-regulated antibody of claim
 1. 11. Amethod of treating a coagulopathy, comprising administering to a patientin need thereof a therapeutically effective amount of theprotease-regulated antibody of claim
 1. 12. The method of claim 11,wherein the coagulopathy is hemophilia.
 13. A method of modulatingprocoagulant activity of an anti-TFPI antibody, comprising adding aprotease cleavage site into the anti-TFPI antibody.
 14. The method ofclaim 13, wherein the protease cleave site is a thrombin cleavage site.15. The method of claim 13, wherein the protease cleavage site is aplasmin cleavage site.
 16. The method of claim 13, wherein the proteasecleavage site is a Factor Xa cleavage site.
 17. A method of promotinggeneration of thrombin or plasmin, comprising contacting TFPI with aprotease-regulated antibody of claim 1.